Natural Products for Drug Discovery: Discovery of Gramines as Novel

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Agricultural and Environmental Chemistry

Natural Products for Drug Discovery: Discovery of Gramines as Novel Agents against a Plant Virus Aidang Lu, Tienan Wang, Hao Hui, Xiaoye Wei, Weihao Chui, Chunlv Zhou, Hongyan Li, Ziwen Wang, Jincheng Guo, Dejun Ma, and Qingmin Wang J. Agric. Food Chem., Just Accepted Manuscript • Publication Date (Web): 07 Feb 2019 Downloaded from http://pubs.acs.org on February 7, 2019

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Journal of Agricultural and Food Chemistry

Natural Products for Drug Discovery: Discovery of Gramines as Novel Agents against a Plant Virus

Aidang Lu†,*, Tienan Wang†, Hao Hui†, Xiaoye Wei†, Weihao Cui†, Chunlv Zhou†, Hongyan Li†, Ziwen Wang‡,*, Jincheng Guo§, Dejun Ma§, Qingmin Wang§,*

†School

of

Chemical

Engineering

and

Technology,

Hebei

University

Hebei Collaborative Innovation Center of Modern Marine Chemical Technology,

of

Technology,

Tianjin

300130,

China ‡Tianjin

Key Laboratory of Structure and Performance for Functional Molecules, MOE Key

Laboratory of Inorganic–Organic Hybrid Functional Material Chemistry, College of Chemistry, Tianjin Normal University, Tianjin Normal University, Tianjin 300387, China; §State

Key Laboratory of Elemento-Organic Chemistry, Research Institute of Elemento-Organic

Chemistry, College of Chemistry, Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Nankai University, Tianjin 300071, China

* To whom correspondence should be addressed. For Aidang Lu, E-mail: [email protected]; Phone:

0086-22-60302812;

Fax:

0086-22-60204274;

For

Ziwen

Wang,

E-mail:

[email protected]; Phone: 0086-22-23766531; Fax: 0086-22-23766531; For Prof. Qingmin Wang, E-mail: [email protected]; Phone: 0086-22-23503952; Fax: 0086-22-23503952.

1

ACS Paragon Plus Environment


1 2 3

ABSTRACT: Plant viral diseases seriously affect crop yield and quality. Natural product

4

gramine (1) and its structure simple analogues 2–35 were synthesized from indoles,

5

amines and aldehydes by one-step. The antiviral effects of these alkaloids were evaluated

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systematically. Most of these compounds were found to have higher antiviral effects than

7

commercial ribavirin for the first time. Especially for compounds 22, 30 and 31 exhibited

8

significantly higher effects than ningnanmycin, since emerged as novel antiviral leads for

9

further optimization. The preliminary implementation indicated that these compounds

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likely inhibit the assembly of TMV by crosslinking TMV CP. Gramine analogues were

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also found to have broad-spectrum fungicidal effects. Although gramine has been

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reported to have influence on germination and development of Erysiphe graminis, these

13

compounds displayed no fungicidal effects against Blum eria graminis f. sp.tritici on

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wheat in our test. Some of these compounds also exhibited certain insecticidal activities.

15 16 17 18

KEYWORDS: natural product, gramine analogues, anti-TMV activity, fungicidal

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activity

20 21 2


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INTRODUCTION

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In the past 60 years, the population has more than doubled. The demand for more

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fast increasing food production is needed.1,2 Plant diseases can cause a significant

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economic and environmental impact on agricultural communities. Tobacco Mosaic Virus

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(TMV), named after its first discovery in tobacco, is a well-studied plant virus. It is

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reported that TMV can infect more than 400 crops including tobacco, pepper, cucumber,

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banana and ornamental flowers.3 Ningnanmycin (Figure 1), perhaps the most effective

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antiviral agent, only gave 50-60% control effects at 500 μg/mL. Ribavirin (Figure 1),

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currently widely used antiviral agent, conveys an anti-TMV effect of less than 50% at

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500 μg/mL. In fact, once a plant is infected with TMV, there is no agent to cure it

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completely. Therefore, it is urgently needed to find agents with novel structures and

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unique mechanisms of action.4

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Nitrogen-containing heterocyclic compounds often exhibit a variety of biological

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activities and are widely concerned in the field of medicine and pesticides. Among them,

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compounds containing indole skeleton have aroused great interest of chemists,5 and have

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been reported to have various kinds of activities, such as antibacterial activity, anticancer

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activity, antiviral activity, protein kinase inhibitory effect.6-12 Gramine (Figure 1) is an

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indole alkaloid widely found in raw plants and in coal tar.12-14 This alkaloid has various

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kinds of activities, such as regulation of bronchial smooth muscle activity, regulation of

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vasodilation activity and controlling blood pressure activity.15 It can control

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mitochondrial energy metabolism in rat liver and beef heart and displayed weak 3



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acetylocholinesterase inhibitory effect.16,17 Recent studies have shown that gramine can

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act as a 5-HT2A receptor and thus exhibits vasodilatory activity.15,18 In agriculture,

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gramine was reported to have influence on germination and development of Erysiphe

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graminis.19 However, there is no report on the antiviral activity of gramine in plants.

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In our previous work, indole alkaloid topsentin A (Figure 1) and its derivatives,20

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were found to have good anti-TMV activities. As another indole-containing natural

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alkaloid, gramine has more simple structure than topsentin A. As a continuation of our

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work, various of gramine analogues were designed (Figure 2), synthesized and

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systematically evaluated for their antiviral, anti-phytopathogenic fungi and insecticidal

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activities.

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MATERIALS AND METHODS

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Instruments. The melting points of the target compounds were determined on an X-4

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binocular microscope (Gongyi Yuhua Instrument Co., China). NMR spectra were

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acquired with a Bruker 400 MHz (100 MHz for

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Chemical shifts were measured relative to residual solvent peaks of CDCl3 (1H: δ = 7.26

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ppm;

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obtained with an FT-ICR MS spectrometer (Ionspec, 7.0 T). Analytical TLC was

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performed on silica gel GF 254. Column chromatographic purification was performed

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using silica gel. The in vitro TMV rod assembly inhibition and 20S CP Disk assembly

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inhibition were tested on transmission electron microscopy (Tecnai G2 F20).

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General Synthesis. The synthetic route is given in Figure 3.

13C:

13C)

instrument at room temperature.

δ = 77.0 ppm) with tetramethylsilane as internal standards. HRMS data were

4


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The detailed operation steps and experimental data of gramines 1–35 can be found in

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the Supporting Information.

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Biological Assay. Each group of tests was repeated three times at 25±1 °C. Activity

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results were given as a percentage scale of 0−100 (0: no activity; 100: total inhibited).

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Detailed bioassay procedures for the anti-TMV,21 fungicidal22-24 and insecticidal22

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activities were described in literature, also can be seen in Supporting Information.

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Mode of Action Studies.

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In vitro TMV rod assembly inhibition ： TMV was purified by Leberman method.25

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TMV RNA purification was performed by RNApure virus kit (CoWin Biosciences) and

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TMV capsid protein (TMV CP) was purified by the acetic acid method.26 Before

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assembling, 20S CP Disk was obtained by hatching CP (20 mg/mL) in pH 7.0 phosphate

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buffer (0.1 M) at 20 °C for 12 h. Then, in vitro TMV assembly experiments were carried

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out by successive adding the phosphate buffer 5 μL (0.1 M, pH 7.0), 20S Disk 4 μL (2

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mg/mL) and TMV RNA 1 μL (200 ng/μL). The mixture was hatched at 20 °C for 12 h

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and could be then transferred into the copper grid for transmission electron microscopy

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(TEM) assay. The assembly reaction mixture (5 μL) was mixed with 0.1 M phosphate

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buffer 5 μL (pH 7.0) and dropped onto the copper film waiting for 5 minutes. After the

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incubation, the droplet was removed by filter paper and negatively stained by 2%

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phosphotungstic acid (pH 7.0) for three minutes. After removing the staining agent, the

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copper was placed at 37 ℃ for 2 h for drying. The morphology of the reconstituted TMV

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rods was imaged at 200 keV on a CCD camera. For the inhibition experiments with the 5



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target compounds, in vitro TMV reconstitution inhibition experiments were performed by

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successive adding phosphate buffer 4.8 μL (0.1 M, pH 7.0), 20S Disk 4 μL (2 mg/mL),

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TMV RNA 1 μL (200 ng/μL) and DMSO 0.2 μL or the target compound (10 μM). Repeat

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each experiment at least three times to ensure the reliability of the data.

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In vitro 20S CP Disk assembly inhibition: For the inhibition tests with the compounds,

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TMV CP was first adjusted with 0.1 M phosphate buffer (pH 7.0) to 20.4 mg/mL. In vitro

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20S CP Disk assembly experiments were carried out by adding 9.8 μL TMV CP (20.4

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mg/mL) and 0.2 μL DMSO or the compound (10 μM). The assembly experiment was

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hatched at 20 °C for 12 h. The morphology of the 20S CP Disk was imaged via TEM at

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200 keV on a CCD camera. Repeat each experiment at least three times to ensure the

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reliability of the data.

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RESULTS AND DISCUSSION

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Chemistry.

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Synthesis of the Compounds 1−35. Indole group was selected as template for structural

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diversification. By introducing a series of functional groups into the side chain, we

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investigated the effects of electronic effect, steric hindrance, hydrophilicity and

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lipophilicity on the activity. The Mannich reaction in the presence of either protic or

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Lewis acids14 is considered as powerful carbon-carbon bond forming processes to afford

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3-substituted indoles. Compounds 1−35 were prepared by using the depicted procedures

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in Figure 3. Substituted indoles were reacted with corresponding aldehydes and amines to

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obtain 1−35 in 53%−97% yields. As the new compounds, 7−9, 12, 14−16, 18 and 24−35 6


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were identified by nuclear magnetic resonance (NMR) and high-resolution mass

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spectrometer (HR-MS). Other known compounds were compared with literature.

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Compound 22 is a known compound, but no physical properties available. So the NMR

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and data of HR-MS of 22 were also given.

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Phytotoxic Activity. The phytotoxic activities of compounds 1−35 were first tested by

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using the test plants which indicated that compounds 1−35 showed no phytotoxic

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activities at 500 μg/mL. No local lesion appear on the plant leaves. The detailed test

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procedures also can be seen in Supporting Information.

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Antiviral Activity. The in vitro and in vivo anti-TMV activities of gramine and its

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analogues were listed in Tables 1 and 2. The commercial plant virucides ribavirin and

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ningnanmycin were selected as the controls.

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In Vitro Anti-TMV Activity. Gramine analogues 1−35 were first tested in vitro

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anti-TMV activities using the conventional half-leaf method.21 As shown in Table 1,

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compounds 2, 4, 8−10, 15, 17, 22, 27, 30, 31 and 35 displayed significantly higher

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antiviral activities than ribavirin. Among the compounds, 22, 30 and 31 exhibited higher

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TMV inhibition effects than ningnanmycin. The mainly difference among 1−4 lies in the

122

substituents in indole ring. Gramine analogues 2−4 displayed significantly higher

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antiviral activities than gramine (1), which showed that the introduction of substituent

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groups at 1-position, 5-position and 6-position of indole ring are favorable for antiviral

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activity. Bromination of 5-position is more favorable than 6-position (antiviral effect: 2 >

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3). Gramine analogues 5−12 displayed higher antiviral activities than gramine (1), further 7



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revealed that the substituents of N,N-dimethylmethanamine region in gramine are

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favorable for antiviral activity. Among analogues 5−12, compounds 8, 9 and 10 exhibited

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much higher antiviral activities than the others, which indicated that the substituents of

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N,N-dimethylmethanamine region in gramine exhibited synergistic effect. The mainly

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difference among 6, 13 and 19−21 lies in the replacement of N,N-dimethyl group in

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gramine. Compound 13 exhibited higher TMV inhibitory effects than the others, which

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showed that the introduction of 2-amine pyridine group is favorable for antiviral activity.

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Among derivatives 13−18, compounds 15 and 17 exhibited much higher antiviral

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activities than the others. Based on the above structure-activity relationships, 5-bromine

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and 1-substituent gramine analogues 22−35 were further designed. Among derivatives

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22−24, compound 22 exhibited much higher TMV inhibitory effects than the others,

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which indicated that the methylation of gramine is favorable for antiviral activity. The

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mainly difference among 25−35 lies in the aryl substituents. Compounds 25, 26, 28, 29,

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32, and 34 showed similar level antiviral activities with ribavirin. Compound 33

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displayed relatively lower antiviral activity than the others. Gramine analogues 27, 30, 31

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and 35 exhibited significantly higher antiviral activities than ribavirin, especially for 30

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and 31 (antiviral effect: 30 and 31 > ningnanmycin).

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In Vivo Anti-TMV Activity. The in vivo antiviral tests including inactivation activity,

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curative activity and protection activity against TMV is carried out using reported

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method.21 As depicted in Table 2, target compounds also showed higher in vivo TMV

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inhibitory effects than ribavirin. Compounds 22, 30 and 31 showed significantly higher 8


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TMV inhibitory effects than ningnanmycin, since emerged as novel antiviral leads for

149

further optimization. Unlike in vitro anti-TMV test results, gramine analogues 1, 4−6, 14,

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15 and 18 exhibited relatively higher in vivo TMV inhibitory effects than in vitro. The

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protection effects of compounds 15, 17 and 30 relatively higher than inactivation effect

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and curative effect, which indicated that compounds 15, 17 and 30 may have a certain

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inducible antiviral activities. The other structure-activity relationships in vivo are similar

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to that of in vitro.

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Preliminary Mode of Action. The preliminary mode of action of compounds 15 and 22

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were evaluated via TEM with RNA inhibitor antofine27 and CP disks assembly inhibitor

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NK020928 as controls. The TMV is 300 nm length rod-shaped particle. It contains a

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single-stranded RNA consisting of about 6400 nucleotides. Its coat protein contains 2130

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protein subunits, each of which is composed of 158 amino acids and spirally arranged

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around RNA molecules.29 The test results revealed that 20S CP Disk and TMV RNA can

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assemble into TMV rod effectively (Figure 4, B). The use of a small amount of DMSO

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does not affect assembly (Figure 4, C). Compounds 15, 22, antofine and NK0209 can

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significantly inhibit the assembly of TMV rod (Figure 4, D−G). Further 20S CP Disk

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assembly inhibition tests were carried out to evaluate the interaction of these compounds

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with TMV CP. The 20S CP Disk can obtained by incubation of TMV CP at 20 °C for 12

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h (Figure 5, A). The use of a small amount of DMSO has no impact on assembly of 20S

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CP Disk (Figure 5, B). As the TMV RNA inhibitor, antofine displayed no impact on 20S

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CP Disk assembly

(Figure 5, C). CP disks assembly inhibitor NK0209 showed 9



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significantly impact on 20S CP Disk. Although the 20S CP Disk can assemble efficiently,

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aggregation and fusion of a large number of CP Disks was detected (Figure 5, D). Just

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like NK0209, compounds 15 and 22 also can effectively induce CP Disks aggregation

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and fusion (Figure 5, E, F). Compound 22 with higher in vivo TMV inhibitory effect than

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15 did exhibit stronger polymerization-promoting ability (As shown in Figure 5, the

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number of 20S CP Disk in F is significantly lower than that in E). The above results

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indicated that these compounds likely inhibit virus assembly by crosslinking TMV CP.

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Fungicidal Activity. Gramines 1−35 were simultaneously determined for fungicidal activity with chlorothalonil and azoxystrobin as controls.

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In Vitro Fungicidal Activity. The in vitro fungicidal effects of gramines 1−35 were

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obtained by using mycelial growth tests22 with 14 plant pathogens as artificial media.

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Gramines 1−35 exhibited broad-spectrum fungicidal activities (Table 3). What’s more,

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these compounds displayed high bioselective. Compounds 17 and 23 displayed about

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similar level antifungal activities against Alternaria solani with chlorothalonil. Most of

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these compounds showed good antifungal activities against Phytophthora capsici and

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Physalospora piricola, especially for compounds 17 and 30 (antifungal activity against

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Phytophthora capsici: 30 > 90%; antifungal activity against Physalospora piricola: 17

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and 30 ≥ 90%). Compound 8 displayed more than 60% antifungal activity against

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Rhizoctonia cerealis.

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In Vivo Fungicidal Activity. Gramines 1−35 were further tested fungicidal activities in

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vivo.22 The pathogens tested in this screen were Sclerotinia sclerotiorum on rape, 10


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Rhizoctonia cerealis on cerealis, Botrytis cinerea on cucumber, Phytophthora capsici on

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capsici, Corynespora cassiicola on cucumber with protection and Blum eria graminis f.

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sp.tritici on wheat. The results (Table 3) showed that lots of compounds displayed more

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than 30% inhibitory effect. Although gramine has been reported to have influence on

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germination and development of Erysiphe graminis,19 all compounds displayed no

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fungicidal activity against Blum eria graminis f. sp.tritici on wheat in our test.

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Insecticidal Activity. Some compounds showed certain insecticidal activity. Compounds

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13, 22 and 32 exhibited broad-spectrum insecticidal activities against Mythimna separate,

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Helicoverpa armigera, Ostrinia nubilalis and Culex pipiens pallens.

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In summary, natural product gramine and its derivatives were prepared and tested for

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their TMV inhibitory effects for the first time. Most of these structure simple compounds

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showed higher TMV inhibitory effects than ribavirin. Compounds 22, 30 and 31 with

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significantly higher TMV inhibitory effects than ningnanmycin emerged as new antiviral

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leads for further research. The introduction of substituents at 1-position, 5-position and

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6-position of indole ring are favorable for TMV inhibitory effect. Bromination of

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5-position is more favorable than 6-position. Compounds 15, 17 and 30 may have a

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certain inducible antiviral activities. The preliminary mode of action studies revealed that

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these compounds likely inhibited TMV assembly by crosslinking TMV CP. Further

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fungicidal activity test showed that these compounds exhibited broad-spectrum fungicidal

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activity. Compounds 17 and 23 displayed about similar level antifungal activities against

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Alternaria solani with chlorothalonil. Insecticidal activity bio-assay showed that gramine 11



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and its derivatives also displayed a certain insecticidal activity. Current study provides

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strong data support for the application of gramines in plant protection.

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ASSOCIATED CONTENT

214

Supporting Information

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The detailed bio-assay procedures. The preparation procedures and the spectra data of

216

compounds 1−35. This material is available free of charge via the Internet at

217

http://pubs.acs.org.

218

AUTHOR INFORMATION

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Corresponding Authors

220

*(A.L.)

221

0086-22-60204274

222

*(Z.W.)

223

0086-22-23766531.

224

*(Q.W.)

225

0086-22-23503952

226

Funding

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This study was supported by Natural Science Fund of China (21772145, 21732002,

228

21672117, 21501042).

229

Notes

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The authors declare no competing financial interest.

231

REFERENCES

E-mail:

E-mail:

E-mail:

[email protected];

Phone:

0086-22-60302812;

Fax:

[email protected];

Phone:

0086-22-23766531;

Fax:

0086-22-23503952.

Fax:

[email protected].

Phone:

12


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(27) Xi, Z.; Zhang, R. Y.; Yu, Z. H.; Ouyang, D. The interaction between tylophorine B and TMV RNA. Bioorg. Med. Chem. Lett. 2006, 16, 4300–4304.

308

(28) Li, X. Y.; Hao, G. F.; Wang, Q. M.; Chen, Z.; Ding, Y.; Yu, L.; Hu, D. Y.; Song, B.

309

A. Ningnanmycin inhibits tobacco mosaic virus virulence by binding directly to its

310

coat protein discs. Oncotarget 2017, 8, 82446–82458.

311 312

(29) Butler, P. J. G. The current picture of the structure and assembly of tobacco mosaic virus. J. Gen. Virol. 1984, 65, 253–279.

Figure Captions 16


Page 16 of 32

Page 17 of 32


Figure 1. Structures of Ningnanmycin, Ribavirin, Gramine and Topsentin A. Figure 2. Design of Gramine Analogues. Figure 3. Synthesis of Gramine Analogues 1–35. Figure 4. TMV Rod Assembly Inhibition of Compounds 15, 22, NK0209 and Antofine. (A) 20S CP Disk (50 nm scale bar); (B) 20S CP Disk + RNA 20S CP Disk + RNA + DMSO

(200 nm scale bar); (D) 20S CP Disk + RNA + antofine

(200 nm scale bar); (E) 20S CP Disk + RNA + NK0209 Disk + RNA + 15

(100 nm scale bar); (C)

(200 nm scale bar); (F) 20S CP

(200 nm scale bar); (G) 20S CP Disk + RNA + 22

(100 nm scale

bar). Figure 5. 20S CP Disk Assembly Inhibition of Compounds 15, 22, NK0209 and Antofine (100 nm scale bar). (A) CP; (B) CP + DMSO; (C) CP + antofine; (D) CP + NK0209; (E) CP + 15; (F) CP + 22.

Table 1. In Vitro Antiviral Activities of Compounds 1−35, Ribavirin and Ningnanmycin 17



Page 18 of 32

against TMV. Compd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Concn (μg/mL)

Inhibition rate (%)a

500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500

23±1 0 51±2 20±2 39±1 5±1 48±2 31±1 33±2 4±1 25±2 0 43±1 16±2 52±2 23±2 49±1 15±1 51±2 19±1 35±2 0 46±1 14±1 36±2 0 35±2 13±1 52±2 17±1 36±1 5±2 50±1 22±2 46±1 13±2 19±2

compd

Concn (μg/mL)

Inhibition rate (%)a

500 16±1 100 0 500 30±2 21 100 0 500 61±2 22 100 27±1 500 35±3 23 100 10±2 500 40±1 24 100 18±2 500 45±1 25 100 18±1 500 39±3 26 100 11±2 500 54±2 27 100 19±1 500 43±2 28 100 16±1 500 46±2 29 100 15±2 500 62±1 30 100 29±1 500 67±2 31 100 31±2 500 44±1 32 100 18±2 500 30±1 33 100 0 500 42±2 34 100 16±2 500 51±1 35 100 20±2 500 41±1 Ribavirin 100 15±2 500 58±2 Ningnanmycin 100 23±2 aAverage of three replicates; All results are 20

18


Page 19 of 32


100

0

expressed as mean ± SD.

Table 2. In Vivo Antiviral Activities of Compounds 1−35, Ribavirin and Ningnanmycin 19



Page 20 of 32

against TMV. Compd 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Concn (μg/mL)

Inactivation effect (%)a

Curative effect (%)a

Protection effect (%)a

500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500 100 500

32±1 0 57±1 29±1 42±2 6±2 59±2 27±1 46±2 12±1 33±2 0 52±1 26±2 57±2 31±1 45±4 7±1 55±1 13±2 43±1 6±1 41±1 6±2.0 36±1 0 44±2 10±1 60±2 13±1 43±2 10±2 56±4 15±2 53±1 29±2 33±2

26±1 0 53±2 22±1 47±2 12±1 53±2 29±1 42±2 7±1 32±2 0 45±1 16±1 53±1 28±1 48±2 9±1 51±1 24±2 36±1 8±1 39±2 11±2 34±2 0 48±1 21±1 55±2 19±2 47±1 18±1 52±2 20±1 51±2 31±1 27±2

37±2 0 51±1 27±1 41±2 8±1 54±2 33±1 39±2 17±2 31±2 0 41±2 9±1 61±1 23±1 41±2 13±1 49±2 27±1 36±2 10±1 34±2 0 38±2 0 42±2 15±1 64±1 32±1 39±2 6±1 60±1 25±1 58±1 25±1 23±2

Compd

Concn (μg/mL)

Inactivation effect (%)a

Curative effect (%)a

Protection effect (%)a

500 21±2 18±2 26±1 100 0 0 0 500 30±2 31±1 30±2 21 100 0 0 6±2 500 64±2 60±1 65±2 22 100 30±2 31±2 35±1 500 32±1 29±2 35±2 23 100 0 0 0 500 41±2 38±1 45±2 24 100 12±2 8±2 13±2 500 48±3 42±1 44±2 25 100 18±2 14±2 16±1 500 40±2 38±1 41±2 26 100 11±2 7±2 16±2 500 57±1 52±2 56±1 27 100 21±2 24±2 23±2 500 47±2 44±3 46±2 28 100 22±2 20±1 27±1 500 45±1 40±2 42±2 29 100 18±2 12±1 16±1 500 61±2 59±1 65±2 30 100 28±1 27±2 33±1 500 68±2 65±1 66±2 31 100 34±2 32±1 35±2 500 44±3 45±1 40±2 32 100 11±2 15±2 12±1 500 32±1 28±1 26±2 33 100 0 0 0 500 41±3 37±3 42±2 34 100 12±2 15±2 17±1 500 52±2 50±3 53±2 35 100 20±1 18±2 23±2 500 38±1 36±2 39±1 Ribavirin 100 11±1 14±1 10±1 500 58 ± 1 56 ± 1 58 ± 2 Ningnanmycin 100 27 ± 2 25 ± 2 30 ± 1 a Average of three replicates; All results are expressed as mean ± SD. 20

20


Page 21 of 32


100

0

0

0

Table 3. In Vitro Fungicidal Activities of Compounds 1–35 and Chlorothalonil against 14 21



Page 22 of 32

Kinds of Fungi. Fungicidal activities (%)a/ 50 μg/mL Compd A.Sb

B.Cb

B.Mb

C.Hb

F.Cb

F.Gb

F.Mb

P.Cb

P.Ib

P.Pb

R.Cb

R.Sb

S.Sb

W.Ab

1

26±1

12±1

6±1

7±2

14±1

27±1

25±1

7±2

14±1

28±1

13±2

12±1

14±2

18±1

2

30±1

19±2

18±1

11±1

22±1

50±1

21±1

52±1

38±1

69±1

42±1

46±2

24±1

33±1

3

35±2

15±2

15±1

11±1

17±1

35±2

25±1

52±1

38±1

19±1

41±1

25±1

19±1

37±2

4

30±1

19±2

15±1

11±1

14±1

31±2

21±2

45±1

19±1

28±1

18±1

12±1

29±2

18±1

5

22±1

20±2

18±1

7±2

22±1

12±2

4±1

24±1

28±2

33±1

34±2

12±1

29±2

37±1

6

30±2

29±1

18±1

15±1

25±1

15±1

8±1

20±1

24±2

58±1

25±1

12±1

24±1

30±1

7

43±1

32±1

15±1

18±1

25±1

12±1

17±2

62±1

14±1

33±2

35±1

46±1

29±1

18±2

8

26±1

7±1

18±1

11±1

22±1

11±2

12±2

17±1

14±1

25±1

63±1

8±1

33±1

26±1

9

30±1

15±1

21±1

18±1

19±2

8±1

8±1

41±1

24±1

58±1

32±1

33±1

33±1

26±2

10

26±1

12±1

18±1

4±1

19±2

19±1

17±1

24±2

14±2

44±1

32±1

21±2

29±2

15±1

11

39±1

12±1

18±1

18±1

22±1

27±1

12±1

62±1

28±2

33±1

41±2

8±1

33±1

19±2

12

43±2

37±2

21±1

33±1

30±1

15±1

25±1

62±2

28±2

69±1

41±1

37±1

33±1

26±2

13

22±2

20±1

21±1

15±1

28±1

8±2

4±1

41±1

24±1

56±2

32±2

4±1

24±1

22±2

14

26±1

12±1

21±1

30±2

25±1

15±1

12±1

17±1

24±1

28±1

35±2

33±1

19±1

33±1

15

13±2

12±1

24±1

18±2

22±1

4±1

12±1

24±1

24±1

53±1

25±1

4±1

14±1

30±2

16

52±1

49±1

33±2

33±1

42±1

38±1

12±1

55±1

47±2

64±1

42±1

46±2

12±1

33±1

17

61±2

58±2

27±2

37±1

39±1

46±1

29±1

45±1

47±2

92±1

35±2

58±1

38±1

30±2

22


Page 23 of 32


18

35±2

42±1

24±1

30±1

28±2

15±2

29±1

62±1

14±1

36±1

31±1

42±1

24±1

26±1

19

17±2

32±1

24±1

18±1

22±1

11±2

12±1

27±1

14±1

50±1

17±2

12±2

14±1

26±1

20

56±2

44±1

36±2

18±1

28±1

39±1

12±1

58±2

62±1

58±1

31±1

46±1

29±1

33±1

21

43±1

37±1

33±2

15±1

25±1

11±1

12±2

42±1

38±1

36±2

21±1

42±1

24±2

30±2

22

35±2

44±1

30±1

19±2

25±2

15±1

13±2

58±1

40±2

64±2

25±1

43±2

33±2

35±1

23

62±2

50±2

37±1

36±2

45±1

38±2

32±2

82±2

53±1

86±2

42±2

43±1

32±2

37±2

24

55±2

46±1

32±2

28±2

35±1

29±2

26±1

75±2

42±1

74±2

38±1

35±1

28±1

29±2

25

16±1

25±2

30±1

14±2

24±2

10±2

12±1

52±2

15±1

53±2

25±1

30±2

22±2

26±2

26

19±2

33±2

28±2

34±2

42±2

19±2

28±2

62±2

28±2

67±1

38±2

42±2

33±2

30±2

27

11±1

16±2

22±1

15±2

26±2

10±1

13±2

58±2

16±1

56±2

21±1

13±2

19±1

26±2

28

23±2

25±1

21±2

19±2

22±1

9±2

11±2

55±1

24±2

46±1

23±1

31±1

11±2

9±1

29

18±2

32±2

25±1

31±2

42±2

18±2

22±1

51±2

26±2

55±2

26±2

34±2

10±1

25±2

30

51±1

45±1

39±2

33±1

42±2

20±1

23±2

92±2

41±1

90±1

30±1

32±2

37±1

39±2

31

10±2

9±1

15±2

33±2

24±2

8±1

15±2

42±2

20±2

38±1

12±2

11±2

23±2

12±2

32

31±1

25±2

28±2

34±1

51±2

21±2

12±2

72±1

32±2

68±2

24±1

23±1

8±2

11±1

33

21±1

15±2

32±2

23±1

31±2

9±2

11±2

62±1

26±2

63±2

21±1

16±2

13±2

14±1

34

26±2

24±1

30±1

25±2

21±1

14±1

13±1

65±2

32±1

59±2

22±1

17±2

9±1

10±2

35

32±2

22±1

32±2

29±2

22±1

16±2

19±2

76±1

38±2

72±2

26±1

19±2

24±2

26±1

Chlorothalonilc

73±2

100

91±1

73±2

100

Natural Products for Drug Discovery: Discovery of Gramines as Novel

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